NOVELTY STATEMENT
We have demonstrated that PCNDs carrying cargo proteins achieve cytosolic protein delivery in an ultrasound-guided manner. To achieve enhanced therapeutic effects and reduced side effects in protein drug treatment, an ultrasound-responsive carrier is promising because focused ultrasound readily penetrates deep into the body in a non-invasive manner and only a limited region is exposed to ultrasound. For endosomal escape of cargo proteins, we employed PCNDs as carriers to destabilize the endosomal membrane via ultrasound-induced vaporization of PCNDs in endosomes. To date, no protein has been reported to be released from endosomes by utilizing PCND vaporization. Here, we designed and synthesized a novel PEG-lipid that can conjugate with any cargo protein on the lipid coating of the droplets through disulfide bond formation because it is required that cargo proteins be released from the carrier in the reductive cytosol. In this study, both cargo proteins with and without free thiols in their wild type forms were released in the cytosol in response to ultrasound by utilizing reversible protein thiolation in the absence of thiol. Thus, our study clearly shows the proof of a principle that PCNDs are versatile carriers for ultrasound-guided cytosolic protein delivery.